KR20110008158A - Compositions and processes for improved mass spectrometry analysis - Google Patents

Abstract

The present invention provides novel additives for improved analysis by mass spectrometry. More specifically, it has been found that ascorbic acid reduces or eliminates the presence of adducts generally present in the mass spectrum. The improved methods and compositions of the present invention can increase accuracy, sensitivity and throughput for samples analyzed by mass spectrometry.

Description

COMPOSITIONS AND PROCESSES FOR IMPROVED MASS SPECTROMETRY ANALYSIS

Related patent application

This patent application is entitled "Composition and Method for Enhanced Mass Spectrometry," US Patent Application No. 12 / 014,671 (2008) by Thomas Becker, designated Representative Document No. SEQ-6015-UT. Filed January 15, 2011). The entirety of this patent application is incorporated herein by reference, including all documents and drawings, under jurisdiction allowing such implementation.

Extinct  Field

The present invention generally relates to compositions and methods for use in mass spectrometry.

Mass spectroscopy is a powerful analytical tool used to determine the molecular weight of analytes in a sample. When using a time-of-flight mass spectrophotometer, the flight speed of ions is about 10 ⁷ times faster than the rate of molecular movement in an electrophoretic gel, so that mass spectrometry provides a fairly rapid method of analysis. Excellent signal-to-noise ratio is realized by repeating the measurement of the spectrum even 10 to 100 times.

Analysis by mass spectrometry typically begins with a sample that is ionized by any means, for example by Matrix Assisted Laser Desorption Ionization (MALDI) or Electrospray (ES) ionization. The MALDI preparation and measurement procedure first consists in inserting analyte molecules onto a sample carrier in a UV-absorbing matrix which is a solid or liquid, IR or usually an organic acid. The sample carrier comprising the matrix and the analyte is placed in an ion source of the mass spectrophotometer. The matrix is vaporized by shortwave laser pulses whereby the analyte molecules are transferred to the gas phase in a non-fragmented state. Analyte molecules are ionized by colliding and reacting with the matrix ions produced simultaneously. A voltage is applied to accelerate the ions into the field-free flight tube. Due to their different mass, ions in the ion source are accelerated at different rates than smaller ions and reach the detector earlier than larger ions. Various times of flight are converted to different masses of ions.

An alternative method of ionizing the analyte is electrospray (or ES). Like MALDI, electrospray can ionize / vaporize polar molecules. First, the sample of interest is dissolved in a solvent present in ionized form over a range. In a typical ES, the solution is then pumped through a thin capillary that lifts to a high potential. Less-filled droplets are injected from the ES capillary into the bath gas at atmospheric pressure and move the pressure and potential gradient down towards the orifice in a mass-spectrophotometer high vacuum system. As the droplets cross this passageway, it is desolvated and reduced in size so that the surface-Columlom force overcomes the surface tension. As a result, the droplets decompose into smaller droplets until ions are desorbed from the droplets or the solvent is completely removed. The exact mechanism of ion formation is not fully understood, but the result is a beam of ions sampled by the mass spectrophotometer. A more detailed description of the ES process is provided in Cole (John Wiley and Sons, New York), published by Electrospray Ionization Mass Spectroscopy: Basics, Instruments and Applications.

Regardless of which ionization method is used, the formation of unwanted adducts during ionization can degrade the quality and resolution of the spectra produced by mass spectrometry. More specifically, the presence of unnecessary adducts may be difficult to detect and analyze analytes, especially low abundance or low mass analytes.

Summary of the Invention

The present invention provides methods and compositions for improved analysis by mass spectroscopy. Provided herein are methods of making mass spectrometric samples that reduce or minimize adduct formation in a cost effective and easy to implement manner that does not otherwise interfere with ionization, mass spectrometry or detection of sample ions. The improved methods and compositions of the present invention can further facilitate the identification and quantification of analyte peaks, thereby increasing the number of correct calls. This improvement proves particularly useful for samples contaminated with sodium and / or ammonia salts, as well as samples with low analyte concentrations. In certain embodiments, ascorbic acid, or a salt, tautomer or analog thereof, as an adduct-reducing addiive that reduces the presence of adducts in the mass spectrum and increases the signal-to-noise (s / n) ratio The use of is described later. While embodiments of the invention are shown below using “ascorbic acid,” those skilled in the art can use ascorbic acid, ascorbate, salts thereof, tautomers thereof, or analogs thereof (hereinafter described) in the methods and compositions described herein. You will know.

In one embodiment, the invention is a method of reducing adduct formation in a sample comprising an analyte to be analyzed by mass spectrometry, the adduct-reducing comprising ascorbic acid prior to analysis of the analyte by mass spectrometry. A method is provided that includes adding an additive to a sample. In related embodiments, the adduct-reducing additive may further comprise ammonium oxalate. In some embodiments, adduct-reducing additives are used in combination with other adduct-reducing additives (known or not yet found adduct-reducing additives). In certain embodiments, adduct-reducing additives are used in combination with one or more resins.

In certain embodiments, the analyte is a nucleic acid such as deoxyribonucleic acid or ribonucleic acid. The improvements described herein can be applied to a variety of mass spectrometry formats because they can produce undesirable adducts in sample handling procedures prior to mass spectrometry. Examples of mass spectrometry formats include, but are not limited to, Matrix-Assisted Laser Desorption / Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry (MS). , Laser Desorption Mass Spectrometry (LDMS), Electrospray (ES) MS, Ion Cyclotron Resonance (ICR) MS, and Fourier Transform MS. The improvements described herein are readily applicable to mass spectrometry formats ("ionized MS", such as MALDI-TOF MS, LDMS, ESMS) in which analytes are volatilized and ionized.

The invention also provides a method for reducing the formation of adducts in a sample comprising analytes to be analyzed by mass spectrometry, wherein the adduct-reducing additive comprising ascorbic acid is added to the matrix prior to analysis of the analytes by mass spectrometry. It provides a method comprising the step of adding. The method may also include an additional step of adding an adduct-reducing additive to the analyte (as well as the matrix) prior to analysis of the analyte by mass spectrometry. In related embodiments, the adduct-reducing additive may further comprise ammonium oxalate. In some embodiments, the matrix composition comprises 3-hydroxypicolinic acid (3-HPA), di-ammonium citrate (DAC), or a combination thereof. In certain embodiments, the analyte is a nucleic acid such as deoxyribonucleic acid or ribonucleic acid. In some embodiments, adduct-reducing additives are also added to the analyte.

In certain embodiments, the invention provides a method of making a substrate suitable for use in mass spectrometry, comprising depositing a matrix material comprising an adduct-reducing additive on a substrate, wherein the adduct is Reducing additives provide a manufacturing method comprising ascorbic acid. In related embodiments, the adduct-reducing additive may further comprise ammonium oxalate. In another related embodiment, the method further comprises sealing the substrate. The method for sealing the substrate includes, by way of non-limiting example, conditions carried out by a packaging method such as vacuum sealing and heat sealing. In some embodiments, the method further comprises treating the substrate with a substance or gas that minimizes oxidation. In certain embodiments, the substrate is washed with an inert gas such as argon before being sealed. In certain embodiments, the substrate is sealed and / or packaged to limit or eliminate exposure to light or UV radiation prior to analysis by mass spectrometry. In some embodiments, the substrate is sealed and / or packaged to maintain a given pH. In certain embodiments, the substrate is packaged in a container including, by way of non-limiting example, a polymer container (eg, polyethylene, polypropylene, polystyrene container). In some embodiments, the substrate comprises silica or silicon dioxide.

The present invention also provides compositions for analysis by mass spectroscopy, including analytes and adduct-reducing additives. In certain embodiments, the adduct-reducing additive comprises ascorbic acid. In related embodiments, the adduct-reducing additive may further comprise ammonium oxalate.

In some embodiments, the present invention provides a composition suitable for analysis by mass spectrometry comprising an analyte and an adduct-reducing additive, wherein the additive comprises ascorbic acid. This composition may also include ammonium oxalate.

In certain embodiments, the present invention provides a target site for mass spectrometry comprising an adduct-reducing additive comprising a substrate and ascorbic acid. This target site may also include ammonium oxalate. In some embodiments, this target site may further comprise a matrix material. In related embodiments, the matrix material is preloaded on the substrate. In certain embodiments, the target site may further comprise an analyte. In some embodiments, the composition is sealed. Methods for sealing a substrate include, by way of non-limiting example, vacuum sealing and heat sealing. In certain embodiments, the composition is treated with a substance or gas to minimize oxidation. In some embodiments, the composition is washed with an inert gas such as argon before being sealed. In some embodiments, the composition is sealed and / or packaged to limit or eliminate exposure to light or UV radiation. In certain embodiments, the composition is sealed and / or packaged to maintain a given pH.

Also provided herein are methods for preparing analytes for mass spectrometry, comprising (a) contacting a solution comprising an analyte with a composition comprising ascorbic acid or a salt, tautomer or analog thereof, for mass spectrometry Preparing a sample; And (b) introducing the sample into the mass spectrophotometer. In certain embodiments, the composition also includes ammonium oxalate. The analyte is sometimes a nucleic acid including, but not limited to, deoxyribonucleic acid, ribonucleic acid, or the like or combinations thereof. In some embodiments, the mass spectrometry is a matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectroscopy, laser desorption mass spectroscopy (LDMS), electrospray (ES) mass spectroscopy, ion cyclotron resonance (ICR) mass spectroscopy, and Fourier transform mass spectroscopy. In some embodiments, the composition comprises ascorbic acid, and in certain embodiments, the composition does not comprise analogs of ascorbic acid.

Also disclosed herein are methods of analyzing analytes by mass spectrometry, comprising: (a) introducing a sample into a mass spectrophotometer, wherein the sample comprises analyte and ascorbic acid, or salts, tautomers or analogs thereof; And (b) analyzing the sample by mass spectroscopy. In some embodiments, the sample further comprises ammonium oxalate. The analyte is sometimes a nucleic acid including, but not limited to, deoxyribonucleic acid, ribonucleic acid, or the like or combinations thereof. In some embodiments, the sample comprises an analyte and ascorbic acid, and in certain embodiments, the sample does not include analogs of ascorbic acid.

Also disclosed herein is a substrate comprising an array of spots, wherein each spot is (i) a matrix for matrix-assisted laser desorption / ionization (MALDI) mass spectrometry and (ii) ascorbic acid, or salts, tautomers or analogs thereof It provides a substrate comprising a. In some embodiments, each spot further comprises ammonium oxalate, and in certain embodiments, one or more of the spots further comprises an analyte. The analyte is sometimes a nucleic acid including, but not limited to, deoxyribonucleic acid, ribonucleic acid, or the like or combinations thereof. In some embodiments, the matrix comprises 3-hydroxypicolinic acid (3-HPA), or another matrix suitable for analyzing nucleic acids by MALDI mass spectrometry. In certain embodiments, the composition of spots comprising ascorbic acid and a matrix (eg, 3-HPA) absorbs ultraviolet light (eg, from about 220 nm to about 300 nm (eg, from about 260 nm to about 270 nm). ; Absorbs UV light at 266 nm). In some embodiments, the matrix is subjected to MALDI mass spectrometry, including, but not limited to, ferulic acid, sinafic acid, alpha-cyano-3-hydroxy-cinnamic acid and alpha-cyano-4-hydroxy-cinnamic acid. By appropriate components to the protein or peptide. In certain embodiments, the substrate is a chip (eg, a silicon chip). In some embodiments, each spot comprises ascorbic acid, and in certain embodiments, each spot does not comprise an analog of ascorbic acid.

Examples of mass spectrometry that can be improved by the methods and compositions of the present invention include, by way of non-limiting example, nucleic acid sequencing, genotyping or methylation analysis. This analysis can be qualitative or quantitative analysis performed by mass spectroscopy.

Brief description of the drawings

1 shows mass spectra generated using 17-leukin synthetic oligonucleotides (GTG GTG GTG GTG GTG GT) directly spotted on an unmodified standard matrix. In this figure, the presence of ammonia adduct is confirmed (peak height is approximately 12% of the parent peak at 5335 Da).

2 shows the mass spectra generated using the same 17-leucine synthetic oligonucleotides (GTG GTG GTG GTG GTG GT). As confirmed in this figure, the ammonia adduct no longer exists.

FIG. 3 shows the mass spectra generated from RhD gene extension products spotted on an unmodified standard matrix that produces an expansion peak at 7571 Da and a +55 Da adduct peak (NH 3 + K) at 7626 Da. Adduct peaks result in false positive insertion calls (SNR: 2; probability: 88%).

4 shows the mass spectra generated from the RhD gene extension product spotted on an ascorbic acid denatured matrix at 7571 Da, but without the +55 Da adduct peak. The positive error insertion call is removed (SNR: 0; probability: 0%).

Detailed description of the invention

Mass spectrometry provides a fairly accurate and sensitive way to determine the molecular weight of analytes such as nucleic acids and peptides. Mass spectrometry thereby provides a powerful tool for analysis that is otherwise difficult to measure molecules. However, the presence of unnecessary adducts can be difficult to accurately detect and analyze analytes, especially low abundance or low mass analytes. This problem is exacerbated when multiple analytes are detected in a single mass spectrum as part of multiple reactions.

Adducts are formed when ions, usually cations, associate with biomolecules under mass spectroscopic conditions, creating unwanted mass peaks. Any portion of sample processing (e.g. biochemistry, sample handling, sample dispensing, etc.), sample cleanup (e.g., resin addition), sample ionization, or sample detection can contribute to adduct formation.

In the case of matrix-assisted laser desorption / ionization (MALDI) mass spectrometry, the matrix material can itself serve as a source of adduct formation. For example, a mixture of 3-hydroxypicolinic acid (3-HPA) and di-ammonium citrate (DAC) is often used as an effective UV-matrix for MALDI-based nucleic acid analysis. Ammonium salts (eg DACs) are especially added to the matrix, and 3-HPA, because they are known to further reduce the cation adduct formation for single stranded DNA (ssDNA) (Wu, JK , et al., Rapid Comm . Mass Spectrom . 1993, 7, 191; Pieles, et al., Nucleic Acid Research , 1993, 21, 14, 3191). However, the results provided herein suggest that DAC is the main source for ammonia (NH ₃ ) adduct formation. For example, FIG. 1 shows the presence of NH ₃ adduct mass peak at +17 Da in the mass spectrum.

Another source of ammonia adducts is ammoniaylated cation exchange resins that can be used to desalting analytes prior to MALDI analysis (Nordhoff, E., et al., Rapid Comm. Mass Spectrom. 1992, 6 , 771). As described in the examples below, the ammonia adduct is formed primarily of guanine and thymine bases. Thus, such adducts are further mentioned in assays in which nucleic acid analytes are enriched in these bases.

In addition, ammoniaylated cation exchange resins cannot completely remove all sodium ions from the backbone of DNA. This lack of removal results in a sodium ion adduct mass peak at +22 Da. Other adducts may be formed from the matrix material and may be more common in the presence of thymine base. For example, when 3-hydroxypicolinic acid is used as the matrix, adduct mass peaks are found at 94, 138 and 188 Da. At the same time, all of these adduct peaks can lead to misinterpretation of the mass spectrometry results. Thus, compositions and methods that significantly reduce the amount and frequency of adducts, particularly alkali and ammonia adducts, are useful for improving the accuracy, sensitivity, and throughput of assays based on mass spectrometry. The presence of the adduct-reducing additive can reduce or discard the adduct peaks present in the sample analyzed without the adduct-reducing additive. As confirmed herein, the addition of ascorbic acid was found to reduce the average adduct formation score (as defined herein) to 40% or less compared to the resin-treated standard matrix at pseudo expanded product strength (Example 1 to 3). In addition, matrices modified with ascorbic acid additives have been shown to be physically and chemically stable over a period of six months by allowing the substrate to be pretreated with ascorbic acid during manufacture (see Example 4).

Analytes

The present invention can enhance assays based on mass spectrometry of analytes such as nucleic acids (eg oligonucleotides and polynucleotides), proteins, peptides and lipids including certain analogs and conjugates such as glycoproteins or lipoproteins . Other substances that can be corrected by MALDI analysis by the teachings herein are small molecules, metabolites, natural products and drugs. The methods and compositions of the present invention are particularly useful for polynucleotides comprising most of guanine and thymine since these polynucleotides are more sensitive to the formation of ammonia adducts.

The term "nucleic acid" as used herein refers to single-stranded and / or double-stranded polynucleotides such as deoxyribonucleic acid (DNA), and ribonucleic acid (RNA), as well as analogs or derivatives of RNA or DNA. Also derivatives of nucleic acids such as peptide nucleic acid (PNA), phosphorothioate DNA, acyclo nucleotides and other analogs and derivatives thereof, or combinations thereof are included within the term "nucleic acid".

Nucleotide analogs included in polynucleotides include, for example, mass modified nucleotides capable of differentiating the mass of the polynucleotides; Nucleotides containing a detectable label capable of detecting the polynucleotide, such as a fluorescent, radioactive, luminescent or chemiluminescent label; Or nucleotides containing reactive groups such as biotin or thiol groups that are easy to immobilize the polynucleotides on the solid support. Polynucleotides may also contain one or more backbone bonds that are selective degradable, such as chemical, enzymatic or photodegradable. For example, a polynucleotide may comprise one or more deoxyribonucleotides, accompanied by one or more ribonucleotides, which may be accompanied by one or more deoxyribonucleotides, and such sequences are cleavable from the ribonucleotide sequence by base hydrolysis. The polynucleotides may also contain chimeric oligonucleotide primers that may include one or more bonds that are relatively resistant to degradation, such as nucleotides linked by peptide nucleic acid bonds and one or more nucleotides at the 3 'end linked by phosphodiester bonds, and the like. And polymerase.

Sample

As used herein, “sample” refers to a composition containing the analyte to be analyzed. In certain embodiments, the sample is from a "biological sample." Biological substances are generally considered to be any substance obtained from a source of life (eg, humans, animals, plants, bacteria, fungi, protozoa, viruses). The biological sample may be in any form, including solid materials (eg, tissues, cell pellets, and biopsies) and biological fluids (eg, urine, blood, saliva, amniotic fluid, and mouthwashes (containing oral cells)). . Preferably, the solid material is mixed with the fluid.

matrix

Matrix materials are used in certain forms such as mass spectroscopy, such as MALDI spectroscopy. The matrix material acts to separate the analyte molecules from each other, absorbing the energy imparted by the laser photons, and transferring this energy to the analyte molecule to induce its desorption and ionization. Once the analyte is ionized, a mass spectrophotometer, such as a time of flight (TOF) analyzer, can be used to measure the ion mass.

The choice of matrix material for mass spectroscopy often depends on the type of biomolecule analyzed. For example, for nucleic acid analysis by mass spectrometry, the matrix used is often a mixture of 3-hydroxypicolinic acid (3-HPA) and di-ammonium citrate (DAC). Another matrix material used to facilitate ionization of the sample analyte is 2,5-dihydroxybenzoic acid (DHB). DHB also has the disadvantages of adduct formation and chemical noise generation that interfere with sample analysis. Alpha-cyano-4-hydroxycinnamic acid (α-CHCA) is an example of a widely used matrix for ionization of protein and peptide analytes in matrix-assisted laser time-of-flight mass spectrometry. However, α-CHCA adducts may interfere with the ability to detect low and mass analytes accurately, even in common and low presence. Additional matrices that can be used as the free radical scavenger additives described herein for improved mass spectroscopy are Li et al. (Rapid Comm. Mass Spectrom. 12: 993-998 (1998), MC Fitzgerald) and LM Smith (Annu). Rev. Biophys. Biomol. Struc. 1995. 24: 1 17-40), and Nordhoff et al. (Mass Spectrometry Reviews, 1996, 15, 67-138; all of which are incorporated herein by reference) Examples include, but are not limited to, 2,4,6-trihydroxyacetophenone (THAP), anthranilic acid, nicotinic acid, salicylamide, 1-isoquinolinol, T-2- (3- (4-t -Butyl-phenyl) -2-methyl-2-propenylidene) malononitrile (DCTB), sinaflic acid (SA), ditranol (DIT), 3-aminoquinoline, trans-3-indoleacrylic acid (IAA) , 2- (4-hydroxyphenylazo) benzoic acid (HABA), succinic acid, 2,6-dihydroxyacetophenone, ferulic acid, caffeic acid, glycerol and nitroaniline.

The matrix material may be combined with the additive (s) or analyte (s) before or after the matrix is deposited on the substrate. When an analyte is inserted into a matrix of light-absorbing material, the matrix is generally present in excess of the analyte. During crystallization it is usually advantageous for the MALDI method to incorporate some form of analyte molecule into the crystalline matrix material or at least between small matrix crystals into the boundary surface. In some embodiments, the additive is first mixed with the matrix material in solution and the combined matrix material / additive solution is deposited on the substrate to be crystallized.

In various embodiments, the matrix is deposited on a substrate to form individual spots by dissolving the matrix in a solution comprising ascorbic acid and a suitable solvent such as water. The resulting solution is deposited on a MALDI substrate and the substrate can be placed in a vacuum chamber such that the matrix / ascorbic acid solution is dried under vacuum. In one embodiment, ascorbic acid acts as matrix material, alone or in combination with other matrices.

Various methods can be used to deposit the matrix, additives or analytes on the substrate. In one embodiment, the application of each element is carried out in a separate step. For example, the matrix material may be preloaded on the substrate and the analyte may be added later using a suitable liquid dispensing device (eg piezoelectric, pin tool dispensing device). In some embodiments, the elements are deposited in combination. For example, the matrix and additives may be combined first (ie, dissolved in a solvent) and deposited together on the substrate, followed by addition of the analyte. In some embodiments, the matrix or matrix / additive deposits may be dried on a substrate to form crystals of the matrix as the solvent evaporates. Subsequent deposition of the analyte solution on top of the dried matrix leads to partial dissolution of the dried matrix deposits by the analyte and co-crystallization of the redissolved matrix.

In certain embodiments, the adduct-reducing additive is used directly as the matrix material, and in some embodiments, the adduct-reducing additive is used as a component of the matrix material. In some embodiments the matrix material is one or more of an adduct-reducing additive and the mass spectrometry matrix material described herein (eg, 3-HPA, DAC, DHB, CHCA, THAP, DCTB, DIT, SA, IAA, HABA). One or more). For embodiments in which the adduct-reducing additive is used as one or more mass spectrometry matrix materials, the adduct-reducing additive may be from 99% to 1% by weight of the total weight of the matrix material (eg, about 5% of the total weight of the matrix material). Wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, 55 wt%, 60 wt%, 65 wt% , 70 wt%, 75 wt%, 80 wt%, 85 wt%, 90 wt% or 95 wt%), and sometimes the adduct-reducing additive is about 1:20, 1:19, 1:18, 1 : 17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1: 9, 1: 8, 1: 7, 1: 6, 1: 5 , 1: 4, 1: 3 or 1: 2, etc. (ie, molar additive to molar mass spectroscopy matrix).

additive

As used herein, the term “adduct-reducing addiive” is a substance added to any one or more of the components or reagents required for analysis by mass spectrometry. Such components or reagents include samples, analytes, matrix materials, substrates, or combinations thereof. In certain embodiments, the adduct-reducing additive is ascorbic acid, or any derivative thereof having substantially the same adduct reduction effect.

In certain embodiments, the adduct-reducing additive is a free radical scavenger. Any free radical scavenger suitable for use in mass spectrometry, and in specific examples suitable for use in mass spectrometry of nucleic acids, may be used. Examples of free radical scavengers include, but are not limited to, ascorbic acid, retinol, tocotrienol, tocopherol, coenzyme Q10, melatonin, lycopene, lutein, alpha-carotene, beta-carotene, zeaxanthin, astaxanthin, Cantaxanthin, flavones (e.g. luteolin, apigenin, tangerine), pavonols (e.g. quercetin, camphorol, myricetin, isolehamnetine, proanthocyanidins), pavanones (e.g. , Naringenin, erythiothiol), isoflavone phytoestrogens (e.g., zenithine, dyedzein, glycidane), stilbenoids (e.g. resveratrol, pterostilbene), anthocyanins (e.g. cyaniding, Delphinidin, malvidin, pelargonidine, peonidine, petunidine), phenolic acids and esters (e.g., ellagic acid, gallic acid, salicylic acid, rosemarine acid, cinnamic acid, chlorogenic acid, chicoric acid, galoteannin, ellagitannin) , Nonfalvonoid phenolic (e.g. curcumin, xanthone, Silymarin, eugenol) and organic antioxidants (eg, citric acid, oxalic acid, phytic acid, lignan, uric acid, N-acetylcysteine). One skilled in the art can readily identify free radical scavengers suitable as additives or matrices for mass spectrometry by routinely testing the scavenger in the analytes, as identified in the Examples herein.

In some embodiments, the additive is substantially free of impurities and thus does not need to be purified. If the adduct-reducing additive is not substantially pure, it can be purified by methods known in the art, such as ion-exchange resin purification, to remove impurities.

The additive may be dissolved in liquid form (eg, dissolved in water) and then deposited on the preloaded matrix or directly on the substrate without the preloaded matrix. Alternatively, the additive may be combined with the matrix prior to being deposited on the substrate. The additive and matrix can be combined to obtain a matrix concentration of 1 to about 20 mg / ml and an additive concentration of about 5 to about 50 mM. The use of insufficient amounts of additives does not significantly reduce the formation of adducts, while the use of too many additives can suppress the parent signal in the mass spectrum. One skilled in the art can optimize the analysis of a particular analyte by determining the appropriate amount of additive without undue experimentation to vary the amount of additive and determine its effect on spectral properties.

In some embodiments, adduct-reducing additives may be used alone or in combination with other materials that reduce or eliminate the presence of unnecessary adducts. Ascorbic acid additives can be combined with other additives that can reduce background noise in the mass spectrum. Other known suitable additives include resins, volatile ammonium salts, in particular volatile monobasic, dibasic or tribasic ammonium salts. The salt additive is preferably not too basic so as not to disturb the sample being analyzed. The additives can be monobasic phosphates and sulfates (eg ammonium monobasic phosphates), and dibasic citrate (eg ammonium dibasic citrate), and tribasic citrate (eg ammonium tribasic citrate).

In certain embodiments, the adduct-reducing additive is ascorbic acid, ascorbate, salts thereof, tautomers thereof, or ascorbic acid analogs (including analog salts and tautomers) having a structure according to the formula:

Where

R ¹ and R ² are independently OH, halogen, R ³ , OR ³ , azido, cyano, CH ₂ R ³ , CHR ³ R ⁴ , SR ³ , NR ³ R ⁴ ;

R ³ and R ⁴ are independently H, alkyl, acetylene or cyano, or optionally substituted aryl carbocyclic ring, aryl heterocyclic ring, non-aryl carbocyclic ring or non-aryl heterocyclic ring. Ascorbic acid analogs as used herein are generally free radical scavenging agents, and the free radical scavenging activity of ascorbic acid analogs can be measured by one skilled in the art (eg, oxidizing agents such as 2,6-dichloro). Titration with phenol-indophenol (DCPIP), iodine, iodate and iodide mixtures or N-bromosuccinimide).

As used herein, the term “optionally substituted” indicates that the particular group (s) described may not have non-hydrogen substituents, or that group (s) may have one or more non-hydrogen substituents. Unless defined otherwise, the total number of such substituents that may be present is the same as the number of H atoms present on the unsubstituted form of the described group. When an optional substituent is bonded via a double bond, such as carbonyl oxygen (= 0), that group occupies two available valences, so that the total number of substituents that can be included is reduced according to the number of available valences.

Ascorbic acid and analogs thereof may have ionizable groups so that they can be prepared as salts. In such cases, it will be understood that with reference to the compound anywhere, one can also use pharmaceutically acceptable salts in the art. Such salts can be acid addition salts, including inorganic or organic acids, or salts can be prepared from inorganic bases or organic bases in the acidic form of the compounds of the invention. Often, the compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases are well known in the art and include, for example, hydrochloric acid, sulfuric acid, hydrobromic acid, acetic acid, lactic acid, citric acid or tartaric acid, and basic salts for forming acid addition salts. Potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines, and the like. Methods for preparing suitable salts are well accomplished in the art. In some cases, the compounds may contain both acidic and basic functional groups, in which case they may have two ionized groups but no net charge.

Ascorbic acid analogs often contain one or more chiral centers. The present invention encompasses stereoisomeric mixtures having varying degrees of chiral purity, including racemic mixtures, as well as each of the isolated stereoisomeric forms. It also includes various diastereomers and tautomers that may be formed. Compounds of the invention may also exist in one or more tautomeric forms; It is understood that the description herein of one tautomer is for convenience only and also includes other tautomers of the identified form.

As used herein, the terms “alkyl”, “alkenyl” and “alkynyl” include straight, branched and cyclic monovalent hydrocarbyl radicals, and combinations thereof, and when they are unsubstituted, only C and Contains only H. Examples include methyl, ethyl, isobutyl, cyclohexyl, cyclopentylethyl, 2-propenyl, 3-butynyl and the like. The total number of carbon atoms in each such group is sometimes described herein, for example represented by 1-10C, or as C1-C10 or C1-10 when the group may contain up to 10 carbon atoms. Can be. Where heteroatoms (typically N, O and S) can substitute carbon atoms as in heteroalkyl groups, for example, the numbers describing the groups are still described as C 1 -C 6, etc. The sum of the number of and the number of heteroatoms included as substitutions for carbon atoms in the backbone of the described ring or chain is indicated.

Typically, the alkyl, alkenyl and alkynyl substituents of the invention contain one 10C (alkyl) or two 1OCs (alkenyl or alkynyl). Preferably, it contains one 8C (alkyl) or two 8C (alkenyl or alkynyl). Sometimes it contains one 4C (alkyl) or two 4C (alkenyl or alkynyl). A single group may comprise one or more types of multiple bonds, or one or more multiple bonds; Such groups are included within the definition of the term "alkenyl" when they contain one or more carbon-carbon double bonds and within the term "alkynyl" when they contain one or more carbon-carbon triple bonds.

Alkyl, alkenyl and alkynyl groups are often optionally substituted such that such substitution is chemically meaningful. Typical substituents include, by way of non-limiting examples, halo, = 0, = N-CN, = N-OR, = NR, OR, NR ₂ , SR, SO ₂ R, SO ₂ NR ₂ , NRSO ₂ R, NRCONR ₂ , NRCOOR, NRCOR, CN, COOR, CONR ₂ , 0OCR, COR and NO 2, each R is independently H, C 1 -C 8 alkyl, C 2 -C 8 heteroalkyl, C 1 -C 8 acyl, C 2 -C 8 heteroacyl, C 2 -C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C10 aryl or C5-C10 heteroaryl, each R is halo, = 0, = N-CN, = N-OR ', = NR', OR ', NR' ₂ , SR ', SO ₂ R', SO ₂ NR ' ₂ , NR'SO ₂ R', NR'CONR ' ₂ , NR'COOR', NR Optionally substituted with 'COR', CN, COOR ', CONR' ₂ , 0OCR ', COR' and NO ₂ , each R 'is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl or C5-C10 heteroaryl. Alkyl, alkenyl and alkynyl groups may also be substituted with C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl or C5-C10 heteroaryl, each of which may be substituted with a substituent appropriate for the particular group.

A "acetylene" substituent is an optionally substituted 2-10C alkynyl group and has the formula -C≡C-Ra, wherein Ra is H or C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2- C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl, C5-C10 heteroaryl, C7-C12 arylalkyl, or C6-C12 Heteroarylalkyl, each Ra group is halo, = 0, = N-CN, = N-OR ', = NR', OR ', NR' ₂ , SR ', SO ₂ R', SO ₂ NR ' ₂ , NR Optionally substituted with one or more substituents selected from 'SO ₂ R', NR'C0NR ' ₂ , NR'COOR', NR'COR ', CN, COOR', C0NR ' ₂ , 0OCR', COR 'and NO ₂ , each R 'is independently H, C1-C6 alkyl, C2-C6 heteroalkyl, C1-C6 acyl, C2-C6 heteroacyl, C6-C10 aryl, C5-C10 heteroaryl, C7-12 arylalkyl, or C6-12 Heteroarylalkyl, each of which is selected from halo, C1-C4 alkyl, C1-C4 heteroalkyl, C1-C6 acyl, C1-C6 heteroacyl, hydroxy, amino, and = 0 Optionally substituted with a group on; Two R's may be linked to form a three to seven membered ring optionally containing up to three heteroatoms selected from N, O and S. In some embodiments, Ra of —C≡C-Ra is H or Me.

"Heteroalkyl", "heteroalkenyl", "heteroalkynyl" and the like are defined similarly to the corresponding hydrocarbyl (alkyl, alkenyl and alkynyl) groups, although the term "hetero" is defined as 1 to 2 in the backbone moiety. A group containing three O, S or N heteroatoms, or a combination thereof; Thus at least one carbon atom of the corresponding alkyl, alkenyl or alkynyl group is substituted with one of the specified heteroatoms to form a heteroalkyl, heteroalkenyl or heteroalkynyl group. Typical and preferred sizes for the heteroformity of alkyl, alkenyl and alkynyl groups are generally the same as those of the corresponding hydrocarbyl groups and substituents that may be present on the heteroformity are the same as described above for the hydrocarbyl groups . For reasons of chemical stability, unless otherwise specified, such groups are understood not to include two or more adjacent heteroatoms, except when the oxo group is present on the N or S, such as a nitro or sulfonyl group.

As used herein, "alkyl" includes cycloalkyl and cycloalkylalkyl groups, while the term "cycloalkyl" may be used herein to describe carbocyclic non-aromatic groups that are linked through a ring carbon atom, and "cyclo Alkylalkyl "may be used to describe carbocyclic non-aromatic groups that are linked to a molecule via an alkyl linking group. Similarly, "heterocyclyl" can be used to describe non-aromatic cyclic groups that contain one or more heteroatoms as ring members and are linked to the molecule via a ring atom, which may be C or N; "Heterocyclylalkyl" can be used to describe groups that are linked to another molecule through a linking group. Suitable sizes and substituents for the cycloalkyl, cycloalkylalkyl, heterocyclyl and heterocyclylalkyl groups are the same as described above for the alkyl groups. As used herein, these terms also include rings containing double bonds or two, unless the ring is aromatic.

As used herein, "acyl" includes a group comprising an alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical bonded to one of the two available valence positions of a carbonyl carbon atom, wherein the heteroacyl is a corresponding group And at least one carbon other than carbonyl carbon has been substituted with a heteroatom selected from N, O and S. Thus, heteroacyl includes, for example, -C (= 0) OR and -C (= 0) NR ₂ as well as -C (= 0) -heteroaryl.

Acyl and heteroacyl groups are bonded to any group or molecule that is bonded through the open valence of a carbonyl carbon atom. Typically this is a C1-C8 acyl group comprising formyl, acetyl, pivaloyl and benzoyl, and a C2-C8 heteroacyl group comprising methoxyacetyl, ethoxycarbonyl and 4-pyridinoyl. Hydrocarbyl groups, aryl groups and heteroforms of such groups, including acyl or heteroacyl groups, generally can be substituted with substituents described herein as appropriate substituents on each of the corresponding components of the acyl or heteroacyl group.

"Aromatic" moiety or "aryl" moiety represents a monocyclic or fused bicyclic moiety having well-known properties of aromaticity; Examples include phenyl and naphthyl. Similarly, "heteroaromatic" and "heteroaryl" refer to such monocyclic or fused bicyclic ring systems containing one or more heteroatoms selected from O, S and N as ring members. Inclusion of heteroatoms allows aromaticity in six-membered rings as well as in five-membered rings. Typical heteroaromatic systems are monocyclic C5-C6 aromatic groups such as pyridyl, pyrimidyl, pyrazinyl, thienyl, furanyl, pyrrolyl, pyrazolyl, thiazolyl, oxazolyl and imidazolyl, and the monocylic C8-C10 bicyclic groups, such as indolyl, benzimidazolyl, indazolyl, benzotriazolyl, isoquinolyl, quine, are fused with one of the click groups to any one of the phenyl ring or heteroaromatic monocyclic group Fused bicyclic moieties formed by forming noyl, benzothiazolyl, benzofuranyl, pyrazolopyridyl, quinazolinyl, quinoxalinyl, cinnaolinyl, and the like. Any monocyclic or fused ring bicyclic system having aromatic character in terms of electron distribution throughout the ring system is included within the definition herein. It also includes bicyclic groups in which at least one ring directly attached to the rest of the molecule has aromatic character. Typically, ring systems contain 5-12 ring member atoms. Preferably, the monocyclic heteroaryl contains 5 to 6 ring members and the bicyclic heteroaryl contains 8 to 10 ring members.

The aryl and heteroaryl moieties may be substituted with a variety of substituents, including C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C5-C12 aryl, C1-C8 acyl and their heteroforms, each of which is May be further substituted by itself; Other substituents for the aryl and heteroaryl moieties include halo, OR, NR ₂ , SR, SO ₂ R, SO ₂ NR ₂ , NRSO ₂ R, NRCONR ₂ , NRCOOR, NRCOR, CN, COOR, CONR ₂ , 0OCR, COR and NO ₂ , each R is independently H, C 1 -C 8 alkyl, C 2 -C 8 heteroalkyl, C 2 -C 8 alkenyl, C 2 -C 8 heteroalkenyl, C 2 -C 8 alkynyl, C 2 -C 8 heteroalkynyl, C6-C10 aryl, C5-C10 heteroaryl, C7-C12 arylalkyl or C6-C12 heteroarylalkyl, each R is optionally substituted as described above for the alkyl group. Substituents on an aryl or heteroaryl group may, of course, be further substituted with groups described herein appropriate for each type of such substituent or each component of the substituent. Thus, for example, arylalkyl substituents may be substituted on the aryl moiety with substituents described herein typical for aryl groups, and further substituted on the alkyl moiety with substituents described herein typical or suitable for alkyl groups.

Similarly, "arylalkyl" and "heteroarylalkyl" are aromatic and heteroaromatics bonded to the point of attachment via linking groups, such as alkylene, including substituted or unsubstituted, saturated or unsaturated, cyclic or acyclic linkages. It represents a ring system. Typically, the linking group is C1-C8 alkyl or hetero form thereof. Such linking groups may also include carbonyl groups, thereby allowing provision of substituents as acyl or heteroacyl moieties. The aryl or heteroaryl ring in the arylalkyl or heteroarylalkyl group may be substituted with the same substituents described above for the aryl group. Preferably, the arylalkyl group comprises an aryl group and a phenyl ring unsubstituted or optionally substituted with a group as defined above in C1-C4 alkylene substituted with one or two C1-C4 alkyl groups or heteroalkyl groups, Wherein the alkyl or heteroalkyl group can optionally be cyclized to form a ring such as cyclopropane, dioxolane or oxacyclopentane. Similarly, heteroarylalkyl groups are preferably optionally substituted with aryl groups and groups described above as usual substituents on C1-C4 alkylene which are unsubstituted or substituted with one or two C1-C4 alkyl groups or heteroalkyl groups. Optionally substituted C5-C6 monocyclic heteroaryl group, or optionally substituted phenyl ring or C5-C6 monocyclic heteroaryl, and C1 unsubstituted or substituted with one or two C1-C4 alkyl or heteroalkyl groups -C4 heteroalkylene, wherein the alkyl or heteroalkyl group can be optionally cyclized to form a ring such as cyclopropane, dioxolane or oxcyclopentane.

When an arylalkyl or heteroarylalkyl group is described as being optionally substituted, the substituents may be present on the alkyl or heteroalkyl moiety, or on the aryl or heteroaryl moiety, of that group. The substituents optionally present on the alkyl or heteroalkyl moiety are generally the same as those described above for the alkyl group; The substituents optionally present on the aryl or heteroaryl moiety are generally the same as described above for the aryl group.

As used herein, an "arylalkyl" group, when unsubstituted, is a hydrocarbyl group and is described by the total number of carbon atoms in the ring and alkylene or similar linking groups. Thus, the benzyl group is a C7-arylalkyl group and phenylethyl is C8-arylalkyl.

"Heteroarylalkyl" as described above is bonded through a linking group and includes an aryl group different from "arylalkyl" in that one or more ring atoms of the aryl moiety or one atom in the linking group is a heteroatom selected from N, O and S It shows the part to do. Heteroarylalkyl groups are described according to the total number of atoms in the ring and combined linkages, which include aryl groups linked through heteroalkyl linkages; Hydrocarbyl linking groups such as heteroaryl groups linked via alkylene; And heteroaryl groups linked through heteroalkyl linking groups. Thus, for example, C7-heteroarylalkyl should include pyridylmethyl, phenoxy and N-pyrrolylmethoxy.

As used herein, "alkylene" refers to a divalent hydrocarbyl group; The reason is that since alkylene is divalent, it can be linked together with two other groups. Usually, the alkylene is represented by-(CH ₂ ) _n- , where n is 1 to 8 and preferably n is 1 to 4, unless otherwise specified, the alkylene may also be substituted with another group, It may have a different length, and no open valence is needed at the opposite end of the chain. Thus, -CH (Me)-and -C (Me) ₂ -may also be represented as alkylene which may be a cyclic group such as cyclopropane-1,1-diyl. When the alkylene group is substituted, substituents include those normally present on the alkyl group described herein.

In general, any heterotype of any alkyl, alkenyl, alkynyl, acyl, or aryl or arylalkyl group or one of those groups contained within a substituent may optionally be substituted by an additional substituent on its own. The nature of such substituents is similar to that described with respect to the original substituents themselves unless the substituents are described otherwise. Thus, for example, when the embodiment of R7 is alkyl, such alkyl may be optionally substituted with the remaining substituents listed as embodiments for R7, which makes a chemical sense and does not encroach on the size limitations provided in alkyl and the like; For example, alkyl or alkyl substituted with alkenyl simply extends the upper limit of carbon atoms for such embodiments and is not included. However, alkyl substituted with aryl, amino, alkoxy, = 0, etc. is included within the scope of the present invention and the atoms of such substituents are not counted in the number used to describe the groups such as alkyl, alkenyl, etc. described. If the number of substituents is not specified, such alkyl, alkenyl, alkynyl, acyl, or aryl groups may each be substituted with multiple substituents depending on their available valences; In particular, any of the above groups may be substituted, for example, with fluorine atoms in any or all of its available valences.

As used herein, “heteroform” refers to a derivative of a group such as alkyl, aryl or acyl, wherein one or more carbon atoms of the designated carbocyclic group is substituted with a heteroatom selected from N, O and S. Thus, the heterotypes of alkyl, alkenyl, alkynyl, acyl, aryl and arylalkyl are heteroalkyl, heteroalkenyl, heteroalkynyl, heteroacyl, heteroaryl and heteroarylalkyl, respectively. It is understood that up to two N, O or S atoms are usually connected sequentially, except when the oxo group is bonded to N or S to form a nitro or sulfonyl group.

As used herein, "halo" includes fluoro, chloro, bromo and iodo. Fluoro and chloro are often preferred. As used herein, "amino" refers to NH ₂ , but when the amino is described as "substituted" or "optionally substituted" the term includes NR'R ", where R 'and R" are each Independently is H, or is an alkyl, alkenyl, alkynyl, acyl, aryl, or arylalkyl group or a heteromorphic form of one of said groups and is an alkyl, alkenyl, alkynyl, acyl, aryl, or arylalkyl group or said One heteroform of one of the groups is each optionally substituted with a substituent described herein as appropriate for the corresponding group. The term also refers to R 'and R "linked together, which may be saturated, unsaturated or aromatic, contain 1-3 heteroatoms independently selected from N, O and S as ring members and are suitable for alkyl groups. Or a form which forms a 3- to 8-membered ring optionally substituted with the described substituents, or when NR′R ″ is an aromatic group, it is optionally substituted with a substituent typically described by a heteroaryl group.

The term "carbocycle" as used herein refers to cyclic compounds containing only carbon atoms in the ring, while "heterocycle" refers to cyclic compounds comprising heteroatoms. Carbocyclic and heterocyclic structures include compounds having monocyclic, bicyclic or plural ring systems. As used herein, the term “heteroatom” refers to any atom that is not carbon or hydrogen, such as nitrogen, oxygen or sulfur. Illustrative examples of heterocycles include, but are not limited to, tetrahydrofuran, 1,3-dioxolane, 2,3-dihydrofuran, pyran, tetrahydropyran, benzofuran, isobenzofuran, 1,3-di Hydroisobenzofuran, isoxazole, 4,5-dihydroisoxazole, piperidine, pyrrolidine, pyrrolidin-2-one, pyrrole, pyridine, pyrimidine, octahydro pyrrolo [3,4 b] Pyridine, piperazine, pyrazine, morpholine, thiomorpholine, imidazole, imidazolidine-2,4-dione, 1,3-dihydrobenzimidazol-2-one, indole, thiazole, benzothiazole , Thiadiazole, thiophene, tetrahydro thiophene-1,1-dioxide, diazepine, triazole, guanidine, diazabicyclo [2.2.1] heptane, 2,5-diazabicyclo [2.2. 1] heptane, 2,3,4,4a, 9,9a hexahydro 1H beta carboline, oxirane, oxetane, tetrahydropyran, dioxane, lactone, aziridine, azetidine, piperidine, lactam And heteroaryl It can hamhal. Other illustrative examples of heteroaryl include, but are not limited to, furan, pyrrole, pyridine, pyrimidine, imidazole, benzimidazole and triazole.

materials

As used herein, “substrate” refers to an insoluble support that deposits and analyzes analytes. The substrate may be, but is not limited to, silica, glass (eg, glass, controlled-pore glass (CPG)), nylon, Wang resin, Merrifield resin, Sephadex, Sepharose, cellulose, magnetic beads , Dynabead, metal surfaces (e.g. steel, gold, silver, aluminum, silicon and copper), plastic materials (e.g. polyethylene, polypropylene, polyamides, polyesters, polyvinylidenedifluoride (PVDF) ) Or pins (eg, flat surfaces with or without plates, such as the synthesis or analysis of combinations in the pits of wafers (eg, silicon wafers) or arrays of pins suitable for beads). Solid supports include, but are not limited to, beads, chips, capillaries, plates, membranes, wafers, combs, fins, wafers including pits, substantially flat surfaces, arrays of pits or nanoliter wells, and other geometries and those skilled in the art. It may exist in any desired form, including forms known to the. Preferred supports are flat surfaces that are designated for receiving or connecting samples at individual locations. Most preferred is a flat surface with a hydrophobic region surrounding the hydrophilic site for receiving, containing or binding the sample. The base material may be inert to the reagents to be used during the procedure, including matrix materials and solvents common to the operation of the device or MALDI mass spectrometry.

On a substrate, the spot of the matrix or matrix / additive or matrix / analyte / additive often has certain properties. Each spot on the substrate may be about 200 μm to about 1 mm in diameter. Spot diameters are often substantially uniform and spot-to-spot diameter variations are often minimal (eg, fluctuations of about 20 μm). Any center-to-center distance for a spot on the substrate useful for mass spectrometry can be used, for example a center-to-center distance of 2.25 mm or 1.125 mm, and the center-to-center distance between spots on the substrate can be Often substantially uniform. Spots on the substrate may have a thickness in the range of about 10 μm to about 100 μm. The thickness of each spot on the gill is often substantially uniform and the thickness variation of the spot-to-spot is often minimal (eg about 30 μm).

Target site

As used herein, the term “target site” refers to a material, such as a matrix material, a matrix material with additives, or a specific place on the substrate where an analyte can be deposited and retained. The substrate may contain one or more target sites, which may be arranged randomly or in an order of arrangement or other pattern. When used in mass spectrometry, such as MALDI analysis, the target site or the resulting site with deposited material is preferably the same as or smaller than the size of the laser spot concentrated on the substrate undergoing desorption. Thus, the target site can be, for example, a well or pit, pin or bead, or a physical barrier disposed on the surface of a solid support, or a combination thereof, such as beads on a chip, chips in a well, and the like. The target site may be physically disposed on the substrate, or may be etched on the surface of the substrate, or may be a “tower” remaining after etching around the site, or physical-chemical parameters such as relative hydrophilicity, hydrophobicity Or any other surface chemistry that retains liquid therein or on it.

Platform

The methods and compositions of the present invention include atmospheric pressure, chemical ionization (APCl), chemical ionization (Cl), electron impact (EI), electrospray ionization (ESl or ES), fast atom bombardment (FAB), enteric desorption / intestinal ionization (FD). / Fl), matrix assisted laser desorption ionization (MALDI), and thermal spray ionization (TSP). In certain embodiments, the ionization source is MALDI or ES.

The methods and compositions of the present invention can be used with any mass spectrometer. There are a number of recently available and well known mass spectrometers including quadrupoles, time-of-flight (TOF) analyzers, magnetic sections, and Fourier transforms and quadrupole ion traps. The analyzer can also be used in tandem (MS-MS) mass spectrophotometers as tandems. In some embodiments, the mass spectrometer is a TOF analyzer.

In certain embodiments, the analytes are analyzed by mass spectrometry and not by spectroscopic methods. In some embodiments, the analyte generally measures the vibrational frequency of a particular molecule and is not analyzed by infrared spectroscopy (eg, Fourier transform infrared spectroscopy) which generally does not include ionization of the analyte and measurement of the ionized analyte. Do not.

The following examples are not limited and illustrate certain embodiments of the invention.

Example  1: Additive to Analyte

In the examples below, the analytes from the Sequenom iPLEX ^™ reaction were mixed with nanoopure water or ascorbic acid additives to determine the effect of the additives on adduct formation. Jurinke, C, Oeth, P., van den Boom, D., MALDI-TOF mass spectrometry: a versatile tool for high-performance DNA analysis. Mol . Biotechnol . 26, 147-164 (2004); And Oeth, P. et al., IPLEX (TM) Assay: Increased Plexing Efficiency and Flexibility for MassARRAY® System through single base primer extension with mass-modified terminators. The plates were treated in parallel after the Sequenom iPLEX ^™ protocol as described in Sequenom Application Note (2005). During the dilution / conditioning treatment step, 9 □ l of analyte solution was mixed with 25 □ l of ultrapure water as a standard protocol, while also mixing other plates and ascorbic acid to produce a final ascorbic acid concentration of 20 mM.

The stock solution of ascorbic acid was desalted with 1 g / ml protonated resin to remove residual cations from ascorbic acid and to rule out any possible inference from ammonia cation exchange resin. Depending on the workflow, the additive may be added directly to the sample after being directly mixed with the analyte or after further dilution. In this particular example, the dilution step was completed with a dilution solution stored in a 50 ml tray at a volume ratio of 1/25 prior to addition to the analyte solution on an automatic liquid handler.

After each dilution / conditioning treatment step, all plates were dispensed at 10 nl / domain with a pre-matrix standard SpectroChip ^™ (30OmM 3-HPA / 25mMDAC) using a Sequenom Nanodispenser and analyzed on a Sequenom MassARRAY® Analyzer Compact.

Conclusion : The use of ascorbic acid led to an 8% higher number of calls for ascorbic acid-treated samples. In ascorbic acid treated samples, sodium and ammonia adducts were suppressed to the limit of detection. This leads to no false false calls due to ammonia and / or sodium adducts for the ascorbic acid treated samples, whereas the ammonia adducts in the water conditioned samples were repeatedly substituted in the heterozygotes (positive error calls). ).

Example  2: additive to matrix

In the examples below, the novel matrix compositions comprising ascorbic acid (AA) confirmed that adduct formation was reduced, improving the quality of the spectrum.

Matrix solution

Matrix combinations are prepared from the following stock solutions and ultrapure water (stock solutions of different matrix components with cation exchange resins according to the functional groups of the component-acids with exchange resins in the form of H +, and ammonia salts with resins in the form of NH4 +). Was:

3-HPA: 35OmM in 30% aqueous acetonitrile.

AA: 1 M in aqueous solution.

DAC: 226 mg 1 Min aqueous solution.

Standard matrices were prepared with 30 mM 3HPA and 25 mM DAC, while new matrices were prepared with 30 mM 3HPA, 20 mM NH ₄ -oxalate and 20 mM ascorbic acid. See Table 1 below. The final matrix was distributed on SpectroChip at 15-20 nl using Gesim Nanoplotter.

Synthetic Oligonucleotide Samples

Two experiments were performed using synthetic oligonucleotides spotted directly on the matrix of Table 1 above. In a first experiment, a 17-leucine synthetic oligonucleotide (GTG GTG GTG GTG GTG GT) was tested on "existing" resin-treated matrices and "new" ascorbic acid-modified matrices. In FIG. 1 (existing matrix), the ammonia adduct was clearly present (peak height was approximately 12% parent peak at 5335 Da), while in FIG. 2 (new matrix) no ammonia adduct was present. The new matrix can alternatively be referred to as "matrix 63C".

In a second experiment, low mass 17-leucine synthetic oligonucleotides (5044 Da) and high mass 28-mer synthetic oligonucleotides (8436 Da) were tested for adduct formation and depurine reactions. In all experiments, the synthetic oligonucleotide analytes were dispensed on the matrix of Table 1 above at 10 nl per spot using Gesim nanoplotter. The results for the low mass and high mass oligonucleotides are found in Tables 2 and 3, respectively.

Matrix comparisons were based on relative adduct and depurine peak height, and probe height and SNR. Adduct formation scores were introduced into the assay taking into account the relative frequency of the pads exceeding the peak score threshold:

Adduct Formation Score =

= (Relative frequency of the pad above the threshold) * (average adduct peak height)

"Pad" and "spot" on the term description are interchangeable. The standard deviation for the mean adduct peak height was low and similar for all matrices and not reported in the table.

For synthetic 17-lens, the average adduct formation score was 13% lower compared to the resin-treated standard matrix on the new matrix (Table 2A), and for the synthetic 28-mer, the average adduct formation score was for the new matrix (Table 3A). ) Was 29% lower compared to the resin-treated standard matrix.

primer  Extended sample

Further experiments were performed using the proven Sequenom genotyping assay for polymorphism in RhD and AMG genes. iPLEX ^TM black and MassARRAY® techniques (Jurinke is incorporated herein by reference, C, Oeth, P., van den Boom, D., MALDI-TOF mass spectrometry:... a versatile tool for high-performance DNA analysis Mol Biotechnol 26 , 147-164 (2004); and Oeth, P. et al., IPLEX ^TM Assay: Increased Plexing Efficiency and Flexibility for MassARRAY® System through single base primer extension with mass-modified terminators.Sequenom Application Note (2005)). The assay was performed. In summary, first amplify the target area around the SNP by PCR. Oligonucleotide primers were then annealed to the PCR product and allele-specifically extended to a single nucleotide using a mixture of four terminator nucleotides and DNA polymerase. Extension products were transferred to miniaturized chip arrays and analyzed by MALDI-TOF mass spectrometry. Measurement of the molecular weight of the extension product clearly identified the SNP allele present in the sample. The peak area ratio of the mass signal was able to assess the relative abundance of alleles in the sample presented.

In this experiment, 20% higher laser energy (compared to the synthetic oligonucleotide experiment above) was used on the 7316 Da AMG primer extension product spotted on the matrix of Table 1. As can be seen from Tables 4 and 5, the average adduct formation score for the extended product dispensed on the new matrix was reduced compared to the standard matrix (40-50% reduction), and the increased laser energy contributed to the adduct formation. There was no negative effect.

To test the effectiveness of the new matrix in the presence of higher amounts of analytes, increased amounts (10, 15 and 20 nl) of 7528 Da AMG primer extension products were analyzed on the matrix of Table 1. See Table 6 below. The average adduct formation score of the new matrix is lower at the analyte volumes of 10 and 15 nl; At 20 nl, the same score (0.6) was observed for both matrices.

Enhanced mass spectrum

The importance of reduced adduct formation to eliminate false positive calls was clearly illustrated in FIGS. 3 and 4, as confirmed by the results for the RHD-4-psi 3-i genotyping assay. The peak at 7626 Da was not inserted as recorded in the standard matrix (FIG. 3), but at 7571 Da there was a 55 Da (NH ₃ + K) adduct for the extension product. This adduct was completely removed from the fresh matrix (Figure 4).

Example  3: Additives to Matrix and Analytes

New ascorbic acid-modified matrices were also tested in combination with ascorbic acid added to the analyte solution (AMG primer extension product at 8289 Da). As confirmed in Tables 2-6 of Examples 1 and 2, ascorbic acid added to the analyte or to the analyte with the standard matrix significantly reduced the ammonia and sodium adducts. See Table 7, below. Ascorbic acid treated analytes on the standard matrix were reduced by 43% (as compared to raw analytes distributed on the untreated matrix) and 57% decreased on ascorbic acid treated analytes distributed on the new matrix.

Example  4: stability test

Over a six month period, four stability tests were conducted to determine the adduct-reducing properties of ascorbic acid over time. The matrix tested over time did not show any decrease in its performance. Instead, the stability of 3-HPA and its additive DAC, NH ₄ -oxalate in combination with ascorbic acid was confirmed with the obtained SNR and consistent adduct formation scores for ammonia and alkali adducts.

The full text of each patent, patent application, publication, and literature referenced herein is incorporated by reference. The citation of said patents, patent applications, publications and documents does not allow any of the foregoing to be appropriate prior art and does not constitute any admission to the date or content of said publication or document.

Modifications can be made with the above teachings without departing from the basic aspects of the invention. Although the present invention has been described in considerable detail with respect to one or more specific embodiments, those skilled in the art will recognize that embodiments specifically disclosed in this application may vary, and such variations and enhancements are within the scope and spirit of the invention. Belong.

The invention described by way of example herein may be practiced in the absence of any element (s) not specifically disclosed herein. Thus, for example, in each example herein, any one of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with the other two terms. The terms and expressions used are used as descriptions of the terms and are not limited thereto, and the use of such terms and expressions does not exclude any equivalent or portion thereof of the identified and described features, and various modifications have been made to the patent. It is possible within the scope of the claimed invention. The term indefinite article “a”, “an” may refer to one or more of the elements being modified, unless the context clearly dictates one or more of the elements to one or more of the elements ( For example, "device" may mean one or more devices). As used herein, the term "about" refers to a value within 10% (ie ± 10%) of the basic parameters, and the use of the term "about" at the beginning of listing of values modifies each value (ie, "about" 1, 2, and 3 "are about 1, about 2, and about 3). For example, the weight of "about 100 g" may include a weight of 90 g to 110 g. Thus, although the invention has been specifically disclosed as representative embodiments and optional features, it will be understood that variations and modifications of the disclosed concepts herein may be preferred by those skilled in the art, and such variations and modifications may be found herein. It is considered to be in range.

Embodiments of the invention are set forth in the following claims.

Claims (46)

A method of reducing the formation of adducts in a sample comprising an analyte to be analyzed by mass spectrometry, wherein the adduct comprising an ascorbic acid, or salts, tautomers or analogs thereof, prior to analyzing the analyte by mass spectrometry Adding an adduct-reducing addiive to the sample. The method of claim 1, wherein the adduct-reducing additive further comprises ammonium oxalate. The method of claim 1, wherein the analyte is a nucleic acid. The method of claim 3, wherein the nucleic acid is deoxyribonucleic acid. The method of claim 3, wherein the nucleic acid is ribonucleic acid. The method of claim 1, wherein the analysis by mass spectroscopy is performed using Matrix-Assisted Laser Desorption / Ionization Time-of-Flight (MALDI-TOF) mass spectroscopy, laser desorption mass spectroscopy (Laser Desorption Mass Spectrometry (LDMS), Electrospray (ES) mass spectroscopy, Ion Cyclotron Resonance (ICR) mass spectroscopy and Fourier Transfrom mass spectroscopy) How. A method of reducing the formation of adducts in a sample comprising an analyte to be analyzed by mass spectrometry, wherein the adduct comprising an ascorbic acid, or salts, tautomers or analogs thereof, prior to analyzing the analyte by mass spectrometry Adding a reducing additive to the matrix. 8. The method of claim 7, further comprising adding an adduct-reducing additive to the analyte prior to analyzing the analyte by mass spectrometry. The method of claim 7 or 8, wherein the adduct-reducing additive further comprises ammonium oxalate. The method of claim 7 or 8, wherein the matrix composition comprises 3-hydroxy picolinic acid (3-HPA). The method of claim 7 or 8, wherein the matrix composition comprises di-ammonium citrate (DAC). The method of claim 7 or 8, wherein the analyte is a nucleic acid. A method of preparing a substrate suitable for use in mass spectrometry, comprising depositing a matrix material comprising an adduct-reducing additive on a substrate, wherein the adduct-reducing additive is ascorbic acid, or a thereof. A process comprising a salt, tautomer or analog. The method of claim 13, further comprising sealing the substrate. The method of claim 13, further comprising treating the substrate with a substance or gas that minimizes oxidation. The method of claim 13, wherein the substrate comprises silica. The method of claim 13, wherein the adduct-reducing additive further comprises ammonium oxalate. A composition to be analyzed by mass spectrometry, comprising an analyte and an adduct-reducing additive. The composition of claim 18, wherein the adduct-reducing agent comprises ascorbic acid, or a salt, tautomer or analog thereof. The composition of claim 19 further comprising ammonium oxalate. A matrix composition, and a composition comprising ascorbic acid, or a salt, tautomer or analog thereof. A composition comprising a matrix composition, ascorbic acid, or a salt, tautomer or analog thereof, and ammonium oxalate. A composition suitable for analysis by mass spectrometry, including an analyte and an adduct-reducing additive, wherein the additive comprises ascorbic acid, or a salt, tautomer or analog thereof, and ammonium oxalate. A target site for mass spectrometry, comprising a substrate and an adduct-reducing additive comprising ascorbic acid, or a salt, tautomer or analog thereof, and ammonium oxalate. The target site of claim 24, further comprising a matrix material. As a method for preparing an analyte for mass spectrometry,
Contacting a solution comprising an analyte with a composition comprising ascorbic acid or a salt, tautomer or analog thereof to prepare a sample for mass spectrometry; And
Introducing the sample into the mass spectrophotometer
≪ / RTI > The method of claim 26, wherein the composition comprises ammonium oxalate. The method of claim 26, wherein the analyte is a nucleic acid. The method of claim 28, wherein the nucleic acid is deoxyribonucleic acid. The method of claim 28, wherein the nucleic acid is ribonucleic acid. 27. The method of claim 26, wherein the mass spectrometry is performed by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectroscopy, laser desorption mass spectroscopy (LDMS), electrospray (ES) mass spectroscopy, ion cyclotron Resonance (ICR) mass spectroscopy and Fourier transform mass spectroscopy is selected from the group consisting of a manufacturing method. As a method of analyzing an analyte by mass spectrometry,
Introducing a sample into the mass spectrophotometer, wherein the sample comprises an analyte and ascorbic acid, or a salt, tautomer or analog thereof; And
Analyzing Samples by Mass Spectroscopy
How to include. The method of claim 32, wherein the sample further comprises ammonium oxalate. The method of claim 32, wherein the analyte is a nucleic acid. The method of claim 34, wherein the nucleic acid is deoxyribonucleic acid. The method of claim 34, wherein the nucleic acid is ribonucleic acid. 33. The method of claim 32, wherein the mass spectrometry is performed by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectroscopy, laser desorption mass spectroscopy (LDMS), electrospray (ES) mass spectroscopy, ion cyclotron Resonance (ICR) mass spectroscopy and Fourier transform mass spectroscopy. A substrate comprising a spot array, wherein each spot comprises (i) a matrix for matrix-assisted laser desorption / ionization (MALDI) mass spectrometry and (ii) ascorbic acid, or salts, tautomers or analogs thereof materials. The substrate of claim 38, wherein each spot further comprises ammonium oxalate. The substrate of claim 38, wherein at least one of the spots further comprises an analyte. The substrate of claim 40, wherein the analyte is a nucleic acid. 42. The substrate of claim 41, wherein the nucleic acid is deoxyribonucleic acid. 42. The substrate of claim 41, wherein the nucleic acid is ribonucleic acid. The substrate of claim 38, wherein the matrix comprises 3-hydroxypicolinic acid. The substrate of claim 38, wherein the substrate is a chip. 46. The substrate of claim 45, wherein the chip is a silicon chip.

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